The following abbreviations are used herein:
BMDC—bone marrow derived dendritic cells
hCG—human chorionic gonadotropin
HhCG—hyperglycosylated hCG
IDO—indoleamine 2,3-dioxygenase
IFN-γ—interferon gamma
Tregs—regulatory T cells
Th17—T helper 17 cells
TGF—transforming growth factor
TF—Thomsen-Friedenreich antigen
uNK—natural killer cells
IL-10 Interleukin 10 VEGF C—Vascular endothelial growth factor C
VEGF C—Vascular endothelial growth factor C
NPS—normal pregnancy serum
PE preeclampsia
PES preeclampsia serum
sEng—soluble endoglin
GPs—gestational pathologies.
Pregnancy is a dynamic process characterized by immune tolerance, angiogenesis and hormonal regulation. Human chorionic gonadotropin (hCG) is reported as detected on the first day of implantation; its levels peak around gestational week 12 and diminish to low levels during the remainder of pregnancy. hCG is believed to exhibit a number of functions in pregnancy, including the promotion of progesterone production, implantation and decidualization, angiogenesis, cytotrophoblast differentiation, and immune cell regulation. With these myriad functions in mind, hCG dysregulation could lead to adverse pregnancy outcomes. Preeclampsia is a condition marked by insufficient trophoblast invasion and maternal spiral artery remodeling and inflammation. Recent studies have reported a link between preeclampsia and immune cell dysregulation, including reduced numbers of uterine and circulating regulatory T cells (Tregs) and natural killer (uNK) cells.
Managing women for preeclampsia is a topic widely addressed in the art. By way of non-limiting example, reference is made to The Diagnosis and Management of Pre-eclampsia and Eclampsia, O'Loughlin et al. (Institute of Obstetricians and Gynaecologists, Royal College of Physicians of Ireland (Version 1.0. Guideline No. 3, 2011).
hCG Variants
hCG is composed of a and P subunits each consisting of a protein backbone with N-linked and O-linked oligosaccharides. It is now believed that there are four distinct variants: hCG, hyperglycosylated hCG (HhCG), the free β-subunit, and pituitary hCG. These four can be further modified by partial degradation of the hCG molecule, nicking of the intact β-subunit, or variation of the attached oligosaccharides.
These variants play different roles in both normal and abnormal pregnancy. HhCG, has complex β-subunit N- and O-linked oligosaccharides structural alterations. HhCG is produced early normally during pregnancy; it does not have high affinity to LH/hCG receptors, yet is reported to promote invasion and growth of cytotrophoblasts by interacting with transforming growth factor (TGF) β receptors. After the initial three to four weeks of pregnancy, the levels of HhCG become very low and hCG (non-hyperglycosylated) is usually the predominant form Recent studies have reported additional variants with distinct sialylated oligosaccharides of the Lewis type pattern on hCG isolated from serum of pregnant women. Differential expression of such carbohydrates is associated with inhibition of E-selectin-mediated homing of leukocytes and may contribute to early pregnancy loss through poor placental-immune interactions. Without being bound by any particular theory, it is believed that in the time between placentation and parturition a dynamic structural conversion of one form of hCG to alternate forms of hCG is choreographed. This suggests that impairment or alterations in hCG glycosylation patterns affect its signaling and biological activities.
hCG, Angiogenesis and Immune Tolerance
The maternal-fetal interface is replete with immune cells which cross-talk with hormonal, endocrine, and angiogenic regulators to program a normal pregnancy outcome. Among immune cell types, regulatory T cells a specialized CD4 T cell subset phenotyped as CD4+/CD25+/Foxp3+, play an important role in protecting the fetus by dampening harmful inflammatory immune responses at the maternal-fetal interface. It has been shown in humans that Treg numbers increase very early in pregnancy, peak during the early second trimester and then begin to decline until they reach pre-pregnancy levels. Tregs have also been described as significant in immune tolerance of the fetus in the mouse pregnancy model. Tregs have been described as following a gestational age-dependent presence in the uterus. Animal studies further indicate that tolerance to paternal antigens may be initiated during mating when seminal fluid and components of semen have been shown to trigger expansion of the Treg cell population. Further, it has been reported that Tregs migrate toward areas of hCG production, indicating that in normal pregnancy, these cells may be attracted to hCG produced by trophoblasts at the maternal-fetal interface ensuring immune tolerance of the fetus. However, if hCG undergoes dysregulation during pregnancy, its control over immune tolerance pathways may be impaired. Interleukin-10 (IL-10) and the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) are two specific immune regulators. Levels of IL-10, an immunosuppressant, reportedly increase in early pregnancy and remain elevated until the onset of labor, possibly regulating maternal immunity and allowing acceptance of the fetal allograft. IL-10 reportedly regulates uNK cell maintenance and further controls their cytotoxic functions in response to pro-inflammatory challenges during pregnancy. Further, decidual Tregs reportedly inhibit immune stimulation of T cells through IL-10 production. It is believed that the temporal expression of IDO regulates the Tregs and prevents them from being converted to pro-inflammatory Th17 cells. hCG reportedly stimulates IL-10 production of murine BMDC. This same study found that treatment of BMDC with hCG and interferon gamma (IFN-γ) increased IDO mRNA production and enzyme activity.
It is noteworthy that hCG is now considered as an angiogenic factor and thus may regulate an endovascular cross-talk between trophoblasts, endothelial cells, and immune cells represented by uNK cells. In animal studies these specialized cells have been reported as playing a role in spiral artery remodeling and trophoblast invasion. Vascular endothelial growth factor C (VEGF C) production by uNK cells is responsible for their non-cytotoxic activity, and that VEGF C producing uNK cells support endovascular processes in vitro. It is possible that the tolerogenic phenotype of uterine NK cells during early decidualization is be influenced by hCG through stimulation of the quiescent angiogenic machinery. Recent studies indicate that the uNK cells are indeed influenced by hCG. One study reported that hCG induces proliferation of human uNK cells, by interacting through the mannose receptor rather than the LH/hCG receptor.
hCG and Preeclampsia
Some reports define preeclampsia as hypertension in a previously normotensive pregnant female and proteinuria after 20 weeks of pregnancy (or gestation). Preeclampsia affects 5-10% of all pregnancies and remains a leading cause of maternal and fetal morbidity and mortality. Related gestational pathologi include Intrauterine growth restriction, eclampsia, gestational hypertension (i.e., hypertension in pregnancy without proteinuria) broadly termed “gestational pathologies” (“GPs”).
Based on clinical presentation, preeclampsia is considered as a late pregnancy disorder. Molecular events leading to its onset seem to occur earlier in pregnancy. Portrayed as a two stage disorder, maternal symptoms of preeclampsia are classified as consequences of pre-clinical placental pathology associated with poor placental perfusion, inflammation, ischemia/hypoxia, and trophoblast damage. Despite the pro-angiogenic role of hCG, little is known about the endovascular interactions of trophoblasts and endothelial cells and its subsequent effects on spiral arteries especially in the presence of different forms of hCG. Recently we reported that injection of preeclampsia serum in pregnant IL-107″ mice results in hypertension and proteinuria. The treatment also led to a perturbed immune cell population at the maternal-fetal interface. Higher hCG levels in preeclampsia serum at term as compared to normal pregnancy serum has been reported. Several studies have reported a decrease in Treg cell population both in the circulation and in placental bed sections in preeclamptic women as compared to those with normal pregnancy. Since IL-10 and hCG are implicated in normal pregnancy outcome, it is tempting to speculate that deficiency in these molecules may predispose to severe preeclampsia pathology. Animal studies from our lab suggest that IL-10 deficient mice are more sensitive to serum- and hypoxia-induced onset of preeclampsia-like features.
All documents cited herein are incorporated by reference in their entirety as if fully set forth herein.
Reference is made to the following:
    1. Cole L A. New discoveries on the biology and detection of human chorionic gonadotropin. Reprod Biol Endocrinol 2009; 7:8.    2. Cole L A. Biological functions of hCG and hCG-related molecules. Reprod Biol Endocrinol 2010:8:102.    3. Meekins, J. W., Pijnenborg, R., Hanssens, M., McFadyen, I. R., van Asshe, A., 1994. A study of placental bed spiral arteries and trophoblast invasion in normal and severe preeclamptic pregnancies. Br J Obstet Gynaecol. 101, 669-674.    4. Toldi, G, Svec P, Vasarhelyi B, Meszaros G, Rigo J, Tulassay T, Treszi, A. Decreased number of FoxP3+ regulatory T cells in preeclampsia. Acta Obstet Gynecol Scand 2008; 87:1229-33.    5. Williams P J, Bulmer J N, Searle R F, Innes B A, Robson S C. Altered decidual leucocyte populations in the placental bed in pre-eclampsia and foetal growth restriction: a comparison with late normal pregnanc. Reproduction 2009; 138:177-184.    6. Stenman U H, Tiitinen A, Alfthan H, Valmu L. The classification, functions and clinical use of different isoforms of HCG. Hum Reprod Update 2006; 12:769-84.    7. de Medeiros S F, Norman R J. Human choriogonadotropin protein core and sugar branches heterogeneity: basic and clinical insights. Hum Reprod Update 2009:15:69-95.    8. Cole L A, Kardana A, Andrade-Gordon P, Gawinowicz M A, Morris J C, Bergert E R, O'Connor J, Birken S. The Heterogeneity of Human Chorionic Gonadotropin (hCG). III. The Occurrence and Biological and Immunological Activities of Nicked hCG. Endocrinology 1991; 129:1559-1567.    9. Butler S A, Cole L A, Chard T, lies R K. Dissociation of human chorionic gonadotropin into its free subunits is dependent on naturally occurring molecular structural variation, sample matrix and storage conditions. Ann Clin Biochem 1998; 35 (Pt 6):754-60.    10. Kovalevskaya G, Kakuma T, Schlatterer J, O'Connor J F. Hyperglycosylated HCG expression in pregnancy: cellular origin and clinical applications. Mol Cell Endocrinol 2007; 260-262:237-43.    11. Elliott M M, Kardana A, Lustbader J W, Cole L A. Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997; 7:15-32.    12. Cole L A. Hyperglycosylated hCG. Placenta 2007; 28:977-86.    13. Jeschke U, Stahn R, Goletz C, Wang X, Briese V, Friese K. hCG in trophoblast tumour cells of the cell line Jeg3 and hCG isolated from amniotic fluid and serum of pregnant women carry oligosaccharides of the sialyl Lewis X and sialyl Lewis a type. Anticancer Res. 2003; 23(2A):1087-1092.    14. Jeschke U, Toth B, Scholz C, Friese K, Makrigiannakis A. Glycoprotein and carbohydrate binding protein expression in the placenta in early pregnancy loss. J Reprod Immunol. 2010; 85:99-105.    15. Stahn R, Goletz S, Stahn R, Wilmanowski R, Wang X, Briese V, Friese K, Jeschke U. Human chorionic gonadotropin (hCG) as inhibitor of E-selectin-mediated cell adhesion. Anticancer Res. 2005; 25:1811-1816.    16. Somerset D A, Zheng Y, Kilby M D, Sansom D M, Drayson M T. Normal human pregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset. Immunology 2004; 1 12:38-43.    17. Aluvihare V R, Kallikourdis M, Betz A G. Regulatory T cells mediate maternal tolerance to the fetus. Nat Immunol. 2004; 5(3):266-71.    18. Robertson S A, Guerin L R, Bromfield J J, Branson K M, Ahlstrom A C, Care A S. Seminal fluid drives expansion of the CD4+CD25+T regulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol Reprod 2009; 80(5):1036-45.    19. Schumacher A, Brachwitz N, Sohr S, Engeland K, Langwisch S, Dolaptchieva M, Alexander T, Taran A, Malfertheiner S F, Costa S D, Zimmermann G, Nitschke C, Volk H D, Alexander H, Gunzer M, Zenclussen A C. Human chorionic gonadotropin attracts regulatory T cells into the fetal-maternal interface during early human pregnancy. J Immunol 2009; 182:5488-97.    20. Hanna N, Hanna I, Hleb M, Wagner E, Dougherty J, Balkundi D, Padbury J, Sharma S. Gestational age-dependent expression of IL-10 and its receptor in human placental tissues and isolated cytotrophoblasts. J Immunol 2000; 164:5721-8.    21. Thaxton J E, Romero R, Sharma S. TLR9 activation coupled to IL-10 deficiency induces adverse pregnancy outcomes. J Immunol. 2009; 183(2):1 144-54.    22. Murphy S P, Hanna N N, Fast L D, Shaw S K, Berg G, Padbury J F, Romero R, Sharma S. Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery. Am J Obstet Gynecol. 2009; 200(3):308.e1-9.    23. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries J E, Roncarolo M G. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997; 389:737-42.    24. Baban B, Chandler P R, Sharma M D, Pihkala J, Koni P A, Munn D H, Mellor A L. IDO activates regulatory T cells and blocks their conversion into Th17-like T cells. J Immunol 2009; 183:2475-83.    25. Wan H, Versnel M A, Cheung W Y, Leenen P J, Khan N A, Benner R, Kiekens R C. Chorionic gonadotropin can enhance innate immunity by stimulating macrophage function. J Leukoc Biol 2007; 82:926-33. 26. Berndt S, Perrier d'Hauterive S, Blacher S, Pequeux C, Lorquet S, Munaut C, Applanat M, Herve M A, Lamande N, Corvol P, van den Brule F,    26. Frankenne F, Poutanen M, Huhtaniemi I, Geenen V, Noel A, Foidart J M. Angiogenic activity of human chorionic gonadotropin through LH receptor activation on endothelial and epithelial cells of the endometrium. FASEB J. 2006; 20(14):2630-2.    27. Herr F, Baal N, Reisinger K, Lorenz A, McKinnon T, Preissner K T, Zygmunt M. HCG in the regulation of placental angiogenesis. Results of an in vitro study. Placenta. 2007 April; 28 Suppl A:S85-93.    28. Cray B A, Esadeg S, Chantakru S, van den Heuvel M, Paffaro V A, He H, Black G P, Ashkar A A, Kiso Y, Zhang J. Update on pathways regulating the activation of uterine Natural Killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus. J Reprod Immunol 2003; 59:175-91.    29. Manaster I, Mandelboim O. The unique properties of uterine NK cells. Am J Reprod Immunol 2010; 63:434-44.    30. Kalkunte S S, Mselle T F, Norris W E, Wira C R, Sentman C L, Sharma S. Vascular endothelial growth factor C facilitates immune tolerance and endovascular activity of human uterine NK cells at the maternal-fetal interface. J Immunol 2009; 1 82:4085-92.    31. Kane N, Kelly R, Saunders P T, Critchley H O. Proliferation of uterine natural killer cells is induced by human chorionic gonadotropin and mediated via the mannose receptor. Endocrinology 2009; 1 50:2882-8.    32. Roberts J M, Hubel C A. Is oxidative stress the link in the two-stage model of preeclampsia. Lancet 1999; 354: 788-789.    33. Kalkunte S, Boij R, Norris W, Friedman J, Lai Z, Kurtis J, Lim K H, Padbury J F, Matthiesen L, Sharma S. Sera from preeclampsia patients elicit symptoms of human disease in mice and provide a basis for an in vitro predictive assay. Am J Pathol. 2010; 177(5):2387-98.    34. Kalkunte S, Nevers T, Norris W, Banerjee P, Fazleabas A, Kuhn C, Jeschke U, Sharma S. Presence of non-functional hCG in preeclampsia and rescue of normal pregnancy by recombinant hCG. Placenta. 2010, 31: A1 26. 35.    35. Sasaki Y, Darmochwal-Kolarz D, Suzuki D, Sakai M, Ito M, Shima T, Shiozaki A, Rolinski J, Saito S. Proportion of peripheral blood and decidual CD4(+) CD25(bright) regulatory T cells in pre-eclampsia. Clin Exp Immunol. 2007; 149:139-45.    36. Santner-Nanan B, Peek M J, Khanam R, Richarts L, Zhu E, Fazekas de St Groth B, Nanan R. Systemic increase in the ratio between Foxp3+ and IL-17-producing CD4+T cells in healthy pregnancy but not in preeclampsia. J Immunol 2009; 183:7023-30.    37. Prins J R, Boelens H M, Heimweg J, Van der Heide S, Dubois A E, Van Oosterhout A J, Erwich J J. Preeclampsia is associated with lower percentages of regulatory T cells in maternal blood. Hypertens Pregnancy 2009; 28:300-11.    38. Lai Z, Kalkunte S, Sharma S. Pregnancy-specific effects of hypoxia: a mouse model for preeclampsia. Am J Reprod Immunol 2009; 61: 398.    39. Lai Z, Kalkunte S, Sharma S. A critical role of IL-10 in modulating hypoxia-induced preeclampsia-like disease in mice. Hypertension. 2011.    40. WO0070094, “Methods For Predicting Pregnancy Outcome In A Subject By hCGg Assay” John O'connor et al.    41. WO201 1 100462/us201 1201 122 “Hyperglycosylated hCG Detection Device,” Albert R. Nazareth et al.    42. U.S. Pub. No 20100129935 “Pregnancy Testing Method,” Sarah Maddison.    43. U.S. Pub. No. 20080241958, “Method for Determining hCG Levels in Fluid Samples,” Hsiao-Ching Yee et al.    44. US Pub. No. 2006010541 1 Method Of Detecting Early Pregnancy At High Accuracy By Measuring hCG And Hyperglycosylated hCG Concentrations Equally by Laurence A Cole.    45. U.S. Pat. No. 7,572,639 Method And Apparatus For Predicting Pregnancy Outcome, Laurence A. Cole et al.”    45. WO0061638 “Prenatal Screening for Down's Syndrome Using Hyperglycosylated Gonadotropin,” Laurence A. Cole et al.    47. Bioassay Techniques for Drug Development, Rahman, et al, (Informa Healthcare (2001)).    48. Assay Development: Fundamentals and Practices, Ge Wu, (Wiley (2010)).    49. Pre-eclampsia: Etiology and Clinical Practice, Lyall et al., (Cambridge University Press (2010)).    50. The Diagnosis and Management of Pre-eclampsia and Eclampsia, O'Loughlin et al. (Institute of Obstetricians and Gynaecologists, Royal College of Physicians of Ireland (Version 1.0. Guideline No. 3, 2011).